Engineering FeoB-Mediated Iron Uptake Enhances Butyrate Fermentation in Clostridium tyrobutyricum

Membrane transporters are pivotal for microbial adaptation to organic-acid stress by coordinating transmembrane exchange and intracellular homeostasis, yet their roles in clostridial butyrate tolerance remain insufficiently defined. In this study, transcriptomic profiling of Clostridium tyrobutyricum under butyrate challenge identified eight significantly upregulated transporter genes. Functional validation showed that overexpression of the ferrous iron transporter genes feoB1 and feoB2 substantially increased Fe²⁺ uptake, improved maintenance of intracellular ROS levels and butyrate homeostasis, and thereby enhanced fermentation robustness. In fed-batch fermentation, ATCC 25755/feoB1B2 produced 67.6 g/L butyrate, significantly exceeding the levels reported for previously engineered strains (46.8-52.2 g/L). Carbon utilization was simultaneously improved, as indicated by a butyrate/acetate ratio of 10.1 and a butyrate yield of 0.43 g/g, corresponding to increases of 102.0% and 10.3%, respectively, relative to the wild type. These results establish engineering of iron uptake and homeostasis as an effective strategy for high-intensity butyrate biomanufacturing, supporting more efficient and low-carbon production of biobased chemicals.